Found ubiquitously in both bacteria and humans, membrane proteins of the adenosine triphosphate (ATP)–binding cassette (ABC) transporter family have been implicated in both antibiotic and cancer-drug resistance. The mechanisms used by these proteins to expel toxins from cells therefore represent key targets for the development of drugs designed to combat the growing problem of multidrug resistance. Toward this end, researchers from The Scripps Research Institute have succeeded in crystallizing MsbA—an ABC transporter protein—together with a substrate (the molecule to be transported) and a hydrolyzed (spent) form of the nucleotide ATP, the transporter's source of chemical energy. The resulting molecular complex is caught at a moment following the transporter's "power stroke," the force-generating part of the transport cycle. This snapshot suggests a mechanism by which the substrate molecule gets flipped head-over-tail from one side of the membrane to the other, on its way out of the cell.

Super Bugs and Wonder Drugs

In the last few decades, mutant strains of bacteria that are resistant to commonly used antibiotics have emerged. Infections by such strains can be treated with alternative drugs, but an even greater threat is emerging: multiple-drug-resistant bacteria that are no longer susceptible to broad categories of antibiotics. For example, certain strains of the bacteria responsible for epidemic dysentery have become resistant to all but a single drug and may soon become completely untreatable. The effort to develop new antibiotics will benefit from an understanding of the mechanisms by which bacteria expel substances that are toxic to them. One such mechanism can be found in MsbA, a protein that "flips" molecules from one side of a cell membrane to the other, where they can then be jettisoned. In human cells, such transporter proteins play an essential protective role by removing harmful toxins. Unfortunately, this protective action can also reduce the efficacy of certain cancer treatments, whose goal is to selectively destroy cancerous cells. The MsbA structure solved by Chang and Reyes could point the way to a new class of drugs that patients would take in conjunction with antibiotic or chemotherapeutic agents to keep the drugs in the cells and increase their efficacy.

ABC transporters are made up of two subunits, each of which contains a transmembrane domain, where specific substrate molecules can be bound, and a nucleotide-binding domain (the ATP-binding "cassette"), which is conserved across all members of the family. Previous studies have shown MsbA to be extremely flexible, occurring in both open and closed conformations. In the open conformation, the two transmembrane domains connect at their extracellular ends to form an inverted V-shaped molecule. In the closed conformation, both the transmembrane and nucleotide-binding domains are closely packed, and a large chamber accessible from inside the cell is formed. It has also been shown that several members of the ABC transporter family work on both lipids and drug molecules, suggesting a common transport mechanism for these compounds, which are characterized by hydrophilic "heads" and hydrophobic "tails." However, despite attempts to model the structural changes of MsbA and other multidrug-resistant ABC transporters, a detailed view of conformational rearrangements during ATP hydrolysis and substrate transport has been elusive.

In this work, the researchers obtained for the first time the structure of an intact ABC transporter (bacterial MsbA from Salmonella typhimurium) in the presence of a substrate (lipopolysaccharide) and a complex of adenosine diphosphate (ADP), inorganic vanadate, and magnesium that mimics the transition state of ATP during hydrolysis. The structure was determined to a resolution of 4.2 Å at ALS Beamline 8.3.1. The results show that the transmembrane domains are tilted 30° relative to the molecular axis, with extensive interdigitation of the helices. Two lipopolysaccharide molecules are bound on the periplasmic (outer-membrane) side, but only one ADP complex is found in the nucleotide-binding domains. A large rotation and translation in the transmembrane domain results in an opening of approximately 15 Å toward the periplasmic end, allowing access to the internal chamber from the periplasm but not from the cytoplasm.

The observations led the researchers to propose a process in which the lipopolysaccharide substrate initially binds near a site in the transmembrane domain (called the elbow helix) with a high affinity for several cationic heavy metals. During the power-stroke step, the hydrophilic heads of the substrate are sequestered within the internal chamber and flipped to the outer membrane by the rigid-body shearing of the transmembrane domains while the hydrophobic tails are dragged through the membrane. The presence of only one ADP complex suggests that the two nucleotide-binding domains act to hydrolyze ATP alternately, while the presence of two lipopolysaccharide molecules suggests that two substrate molecules may be transported per power stroke.

While much work lies ahead before a complete mechanistic model of substrate transport can be achieved, this model (together with previously solved MsbA structures) provides a framework for interpreting functional data concerning ABC "flippases" that confer multidrug resistance to cancer cells and infectious microorganisms.

Research conducted by C.L. Reyes and G. Chang (The Scripps Research Institute).

Research funding: National Institutes of Health, National Science Foundation, Beckman Young Investigators Grant, Fannie E. Rippel Foundation, Baxter Foundation, and The Skaggs Institute for Chemical Biology. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: C.L. Reyes and G.A. Chang, "Structure of the ABC transporter MsbA in complex with ADP·vanadate and lipopolysaccharide," Science308, 1028 (2005).